Discosoma red fluorescent proteins

ABSTRACT

The present invention relates to directed protein evolution in mammalian cells and improved mutants of Discosoma sp. red fluorescent proteins.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. Application No. 10/152,296,filed May 20, 2002, now U.S. Pat. No. 6,723,537, which claims thebenefit of U.S. Provisional Application No. 60/291,871, filed May 18,2001.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable.

FIELD OF THE INVENTION

The present invention relates to directed protein evolution in mammaliancells and improved mutants of Discosoma sp. red fluorescent proteins.

BACKGROUND OF THE INVENTION

Red fluorescent protein has been isolated from a Discosoma sp. andsequenced (see, e.g., Matz et al., Nature Biotech. 17:969–973 (1999),Gross et al., Proc. Nat'l Acad. Sci. USA 97:11990–11995 (2000)). Avariant with humanized codons has also been engineered (Clontech,“DsRED™”). The crystal structure of red fluorescent protein has beenelucidated, which demonstrated that red fluorescent protein is atetrameric protein (Wall et al., Nat. Struc. Biol. 7:1089 (2000);Yarbrough et al., Proc. Nat'l Acad. Sci USA 16:462–467 (2000)).

Red fluorescent protein (RFP) and DsRED, as well as other fluorescentproteins such as YFP, or GFP from Aequorea victoria, Renilla reniformis,Renilla muelleri, and Ptilosarcus gurneyi, are useful are reportermolecules for a variety of bioassays, including those that use FACS as aselection mechanism (see, e.g., Tsein, Nature Biotechnology 17:956(1999); Tsein, Ann. Rev. Biochem. 6:509–544 (1998); Heim et al., Nature373:663–664 (1995); Heim et al., Proc. Nat'l Acad. Sci. USA 91:1250(1994); Prasher et al., Gene 111:229 (1992); Prasher et al., Trends inGenetics 11:320 (1995); Chalfie et al., Science 263:802 (1994); and WO95/21191). However, brighter, faster folding, and higher expressingvariants would be useful.

Such variants can be made, e.g., using methods of gene shuffling andmutagenesis (see, e.g., U.S. Pat. No. 5,811,238; WO 00/73433; WO00/22115; WO 99/41369; WO 01/04287; WO 00/46344; WO 99/45143, WO99/41368; and Ichiro et al, Protein Science 8:731–740 (1999)). However,the use of such methods for production of variant proteins such asDiscosoma red fluorescent protein variants is not always successful(see, e.g., Baird et al., Proc. Nat'l Acad. Sci. USA 97:11984–11989(2000)). Novel methods of making such variants would therefore beuseful.

SUMMARY OF THE INVENTION

The present invention therefore provides variants of Discosoma redfluorescent protein that have been generated using directed molecularevolution in mammalian cells. The variants of the invention have greatlyimproved brightness, expression, and/or folding kinetics as compared towild type or a codon optimized variant. The present invention alsoprovides novel methods of directed protein evolution in mammalian cellsusing retroviral gene transfer and FACS sorting. Such methods can beused to provide improved variants of fluorescent proteins such asDiscosoma red fluorescent protein and fluorescent proteins from othersources, such as Aequorea victoria, Renilla reniformis, Renillamuelleri, and Ptilosarcus gurneyi.

In one aspect, the present invention provides an isolated Discosoma redfluorescent protein, the protein comprising an amino acid sequence asshown in FIG. 1 with one or more point mutations at an amino acidposition selected from the group consisting of N24, F125, K164, andM183.

In one embodiment, the protein comprises two, three, or four pointmutations at an amino acid position selected from the group consistingof N24, F125, K164, and M183.

In one embodiment, the point mutation at amino acid position N24 is aserine, arginine, or histidine substitution. In another embodiment, thepoint mutation at amino acid position F125 is a leucine or valinesubstitution. In another embodiment, the point mutation at amino acidposition K164 is a methionine substitution. In another embodiment, thepoint mutation at amino acid position M183 is a lysine or threoninesubstitution.

In one embodiment, the protein comprises an amino acid sequence as shownin FIG. 1 with a leucine or valine substitution at amino acid positionF125 and a lysine substitution at amino acid position M183.In anotherembodiment, the protein comprises an amino acid sequence as shown inFIG. 1 with a leucine substitution at amino acid position F125 and alysine substitution at amino acid position M183.In another embodiment,the protein comprises an amino acid sequence as shown in FIG. 1 with avaline substitution at amino acid position F125 and a lysinesubstitution at amino acid position M183.

In one embodiment, the protein comprises an amino acid sequence as shownin FIG. 1 with a leucine or valine substitution at amino acid positionF125 and a serine, arginine, or histidine substitution at amino acidposition N24.In another embodiment, the protein comprises an amino acidsequence as shown in FIG. 1 with a leucine substitution at amino acidposition F125 and a serine substitution at amino acid position N24.

In one embodiment, the protein comprises an amino acid sequence as shownin FIG. 1 with a leucine or valine substitution at amino acid positionF125, a serine, arginine, or histidine substitution at amino acidposition N24, and a lysine substitution at amino acid position M183.Inanother embodiment, the protein comprises an amino acid sequence asshown in FIG. 1 with a leucine substitution at amino acid position F125,a serine substitution at amino acid position N24, and a lysinesubstitution at amino acid position M183.

In one embodiment, the protein comprises an amino acid sequence as shownin FIG. 1 with a methionine substitution at amino acid position K164.

In one embodiment, the protein comprises an amino acid sequence as shownin FIG. 1 with a leucine substitution at amino acid position F125.

In one embodiment, the protein further comprises one or more pointmutations at an amino acid position selected from the group consistingof K93, R18, K139, E149, and D170. In another embodiment, the pointmutation at amino acid position K93 is an arginine substitution. Inanother embodiment, the point mutation at amino acid position R18 is ahistidine substitution. In another embodiment, the point mutation atamino acid position E149 is an aspartic acid substitution. In anotherembodiment, the point mutation at amino acid position D170 is a glycinesubstitution.

In one embodiment, the protein comprises an amino acid sequence as shownin FIG. 1 with a leucine substitution at amino acid position F125, aserine substitution at amino acid position N24, a lysine substitution atamino acid position M183, and a histidine substitution at amino acidposition R18.

In one embodiment, the protein comprises an amino acid sequence as shownin FIG. 1 with a leucine substitution at amino acid position F125, aaspartic acid substitution at amino acid position E149, and a glycinesubstitution at amino acid position D170.

In one aspect, the present invention provides a Discosoma redfluorescent protein that is a fusion protein.

In another aspect, the present invention provides a nucleic acidencoding the Discosoma red fluorescent protein of the invention. In oneembodiment, the nucleic acid is codon-optimized for mammalianexpression. In another embodiment, the nucleic acid encodes a fusionprotein.

In another aspect, the present invention provides a vector comprising anucleic acid encoding the Discosoma red fluorescent protein of theinvention. In one embodiment, the vector is a retroviral vector.

In another aspect, the present invention provides a host cell comprisingthe vector of the invention.

In another aspect, the present invention provides a retroviral cDNAexpression library comprising a nucleic acid encoding the Discosoma redfluorescent protein.

In another aspect, the present invention provides a method of making aprotein variant, the method comprising the steps of: (i) mutating aselected nucleotide sequence encoding a fluorescent protein; (ii)cloning the mutated sequences into an expression vector; (iii)transfecting mammalian cells with the expression vector; and (iv)identifying the variants.

In one embodiment, the protein is a fluorescent protein and variants areidentified by FACS analysis. In another embodiment, the selectednucleotide sequence encodes a fluorescent protein from Discosoma “red”sp., Aequorea victoria, Renilla reniformis, Renilla muelleri, orPtilosarcus gurneyi.

In one embodiment, the selected nucleotide sequence is mutated usingerror prone PCR.

In another embodiment, the expression vector is a retroviral expressionvector.

In another aspect, the present invention provides a method of making afluorescent protein variant, the method comprising the steps of: (i)mutating by error prone PCR a selected nucleotide sequence encoding afluorescent protein; (ii) cloning the mutated sequences into aretroviral expression vector; (iii) transfecting mammalian cells withthe expression vector; and (iv) selecting variants using FACS analysis.

In one embodiment, the selected nucleotide sequence encodes afluorescent protein from Discosoma “red” sp., Aequorea victoria, Renillareniformis, Renilla muelleri, or Ptilosarcus gurneyi.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides the amino acid (SEQ ID NO:2) and nucleotide sequence(SEQ ID NO:1) of a mammalian codon optimized Discosoma red fluorescentprotein. This figure also indicates preferred point mutations in theamino acid sequence for variants.

FIG. 2 provides examples of brighter Discosoma red fluorescent proteinvariants.

FIG. 3 provides a list of mutated Discosoma red fluorescent proteinsisolated using the mammalian directed evolution methods of theinvention.

FIG. 4 shows excitation and emission spectra of certain mutants of theinvention.

FIG. 5 shows a diagram of methods for directed evolution of proteins inmammalian cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides variants of Discosoma red fluorescentprotein, which have enhanced brightness, expression, and/or foldingkinetics. These improved characteristics are useful for functionalscreens as a reporter for gene transcription (e.g., as a fusionprotein), for target characterization and localization of fusionproteins, and for scaffolds for protein and peptide libraries. Forexample, variants of the invention can be cloned into expression vectorsthat are used to express cDNA or random peptide libraries. The variantis positioned in the vector such that it forms a fusion protein with theexpressed cDNA or peptide. The cDNA library can comprise sense,antisense, full length, and truncated cDNAs. The peptide library isencoded by nucleic acids. cDNA libraries are made from any suitable RNAsource. Libraries encoding random peptides are made according totechniques well known to those of skill in the art (see, e.g., U.S. Pat.Nos. 6,153,380, 6,114,111, and 6,180,343). Any suitable vector can beused for the cDNA and peptide libraries, including, e.g., retroviralvectors. The Discosoma variant can thus be used as a selectable marker.

Red fluorescent protein is generally useful for screens employing FACSassays. Red fluorescent protein is also useful in screens for reportergene transcription, fusion protein localization, yeast two hybridexperiments, immunoprecipitation and proteomics, increased affinity ofreceptors for fluorescently labeled ligands, proteins which increase theexpression level of a second protein, altered immunogenicity forfluorescently labeled antibodies, changes in cell shape and size,changes in proton pump activity, relative DNA content in cell cycle andapoptosis, cellular localization and changes in metabolic rates ofcalcium flux, cell division, mitochondrial activity, pH, and freeradical production. Such assays are useful for identifying proteinsinvolved in the cell cycle, cellular proliferation, lymphocyteactivation, ubiquitination pathways, cancer, mast cell degranulation,viral replication and translation (e.g., HCV) and angiogenesis. Inaddition to red fluorescent protein, such screens can also use one ormore additional fluorescent protein, such as Aequorea victoria GFP,Zoanthus YFP and GFP, Aneomonia CFP, Clavularia CFP, D. striata CFP,Renilla muelleri GFP, Renilla reniformis GFP, and Ptilosarcus gurneyiGFP, and variants thereof.

In the present invention, novel methods of directed protein evolutionwere used to obtain improved variants of red fluorescent protein, aswell as other proteins, including other fluorescent proteins asdescribed above. In the methods of the invention, error prone PCR isused to randomly mutagenize a nucleic acid sequence encoding a proteinof interest (see, e.g., Leung et al., Techniques 1:11–15 (1989); Calwell& Joyce, PCR Methods and Applications, 2:28–33 (1992); and Gramm et al.,Proc. Nat'l Acad. Sci. USA 89:3576–3580 (1992)). The inherently lowfidelity of Taq polymerase or other thermostable polymerases can befurther decreased by the addition of Mn+, increasing the Mg2+concentration, and using unequal dNTP concentrations. A preferred methodof EP-PCR is described in Calwell & Joyce, PCR Methods and Applications,2:28–33 (1992) and in Current Protocols, supra. Alternatively, otherwell know mutagenesis methods such as gene shuffling could be employed(see, e.g., U.S. Pat. No. 5,811,238, WO 99/41369, WO 99/41368, and WO00/46344). The library of variant nucleic acids is then transferred tomammalian cells (e.g., Jurkat, A549, Phoenix A, or BJAB) usingretroviral vectors. Variants are detected by any suitable assay, e.g.,in the case a fluorescent protein, by FACS. Clones of interest are thenrescued and isolated. As described in FIG. 1, this technique was used toidentify four preferred sites of point mutations (amino acidsubstitutions) that lead to red fluorescent proteins with enhancedbrightness, altered emission, higher expression, and/or enhanced foldingkinetics (3° or 4° structure).

Definitions

The term “point mutation” refers to a deletion, addition, orsubstitution at a designed amino acid position in an amino acid ornucleotide sequence. Preferably, the term refers to an amino acidsubstitution.

“Discosoma red fluorescent protein” refers to a wild-type proteinisolated from Discosoma species “red” (described and sequenced in Matzet al., Nature Biotechnology 17:969–973 (1999)), as well as a mammaliancodon-optimized variant shown in FIG. 1.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in single- or double-stranded form, or complementsthereof. The term encompasses nucleic acids containing known nucleotideanalogs or modified backbone residues or linkages, which are synthetic,naturally occurring, and non-naturally occurring, which have similarbinding properties as the reference nucleic acid, and which aremetabolized in a manner similar to the reference nucleotides. Examplesof such analogs include, without limitation, phosphorothioates,phosphoramidates, methyl phosphonates, chiral-methyl phosphonates,2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs). Nucleic acidsalso include complementary nucleic acids.

Unless otherwise indicated, a particular nucleic acid sequence alsoimplicitly encompasses conservatively modified variants thereof (e.g.,degenerate codon substitutions) and complementary sequences, as well asthe sequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605–2608(1985); Rossolini et al., Mol. Cell. Probes 8:91–98 (1994)). The termnucleic acid is used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide.

A particular nucleic acid sequence also implicitly encompasses “splicevariants.” Similarly, a particular protein encoded by a nucleic acidimplicitly encompasses any protein encoded by a splice variant of thatnucleic acid. “Splice variants,” as the name suggests, are products ofalternative splicing of a gene. After transcription, an initial nucleicacid transcript may be spliced such that different (alternate) nucleicacid splice products encode different polypeptides. Mechanisms for theproduction of splice variants vary, but include alternate splicing ofexons. Alternate polypeptides derived from the same nucleic acid byread-through transcription are also encompassed by this definition. Anyproducts of a splicing reaction, including recombinant forms of thesplice products, are included in this definition.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetics refers tochemical compounds that have a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence withrespect to the expression product, but not with respect to actual probesequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, for example, recombinant cells express genes that arenot found within the native (non-recombinant) form of the cell orexpress native genes that are otherwise abnormally expressed, underexpressed or not expressed at all.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not found in the same relationship to each otherin nature. For instance, the nucleic acid is typically recombinantlyproduced, having two or more sequences from unrelated genes arranged tomake a new functional nucleic acid, e.g., a promoter from one source anda coding region from another source. Similarly, a heterologous proteinindicates that the protein comprises two or more subsequences that arenot found in the same relationship to each other in nature (e.g., afusion protein).

A “fluorescent” label may be detected by exciting the fluorochrome withthe appropriate wavelength of light and detecting the resultingfluorescence. The fluorescence may be detected visually, or by the useof electronic detectors such as charge coupled devices (CCDs) orphotomultipliers and the like. Similarly, enzymatic labels may bedetected by providing the appropriate substrates for the enzyme anddetecting the resulting reaction product. FACS analysis is a preferredmethod of detection when the label is in a cell.

EXAMPLES

The following example is provided by way of illustration only and not byway of limitation. Those of skill in the art will readily recognize avariety of noncritical parameters that could be changed or modified toyield essentially similar results.

Example 1

Error Prone PCR and Directed Evolution of Discosoma Red SpeciesFluorescent Proteins in Mammalian Cells

To mutagenize Discosoma red fluorescent protein, a mammalian codonoptimized variant (see FIG. 1) was cloned with a flag tag andmutagenized using error prone PCR according to methods known to those ofskill in the art (see, e.g., Current Protocols in Molecular Biology,volume 1, unit 8.3 (Ausubel et al., eds, 1994); Saiki et al., Science239:487 (1988); Leung et al., Technique 1:11–15 (1989); Caldwell &Joyce, PCR Methods and Applications 2:28–33 (1992); and Gramm et al.,Proc. Nat'l Acad. Sci. USA 89:3576–3580 (1992)).

The resulting library of mutagenized sequences was cloned into aretroviral vector expression library using RT-PCR and the retrovirallibrary was used to infect human cells (BJAB cells). The cells weresorted for brighter fluorescence, higher expression, or shiftedemission. Selected clones were isolated using RT-PCR, and sub-librarieswere constructed and selected further with FACS (see FIG. 5). Singlecell clones were isolated and sequenced.

FIG. 2 lists some of the brighter mutants identified in the screen(note: amino acid sequences are off by one from the sequence numberingdescribed in FIG. 1 as the methionine was counted as zero for thepurposes of the numbering in FIG. 2). FIG. 1 lists certain preferredmutations at amino acid positions N24, F125, K164 and M183, e.g.,N24S/R/H; K125L/V; K164M; and M183K. These mutations can exist in themutated variants alone or in any combination of one , two , three, orfour, or optionally with additional point mutations at K93, R18, K139,E149, and D170, e.g., K93R, R18H, E149D, and D170G.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. An isolated Discosoma red fluorescent protein, the protein comprisingthe amino acid sequence shown in SEQ ID NO:2 with one or more pointmutations at an amino acid position selected from the group consistingof N24, F125, K164, and M183.
 2. The protein of claim 1, wherein theprotein comprises two, three, or four point mutations at amino acidpositions selected from the group consisting ofN24, F125, K164, andM183.
 3. The protein of claim 1, wherein the point mutation at aminoacid position N24 is a serine, arginine, or histidine substitution. 4.The protein of claim 1, wherein the point mutation at amino acidposition F125 is a leucine or valine substitution.
 5. The protein ofclaim 1, wherein the point mutation at amino acid position K164 is amethionine substitution.
 6. The protein of claim 1, wherein the pointmutation at amino acid position M183 is a lysine or threoninesubstitution.
 7. The protein of claim 1, wherein the protein comprisesthe amino acid sequence shown in SEQ ID NO:2 with a leucine or valinesubstitution at amino acid position F125 and a lysine substitution atamino acid position M183.
 8. The protein of claim 1, wherein the proteincomprises the amino acid sequence shown in SEQ ID NO:2 with a leucinesubstitution at amino acid position F125 and a lysine substitution atamino acid position M183.
 9. The protein of claim 1, wherein the proteincomprises the amino acid sequence shown in SEQ ID NO:2 with a valinesubstitution at amino acid position F125 and a lysine substitution atamino acid position M183.
 10. The protein of claim 1, wherein theprotein comprises the amino acid sequence shown in SEQ ID NO:2 with aleucine or valine substitution at amino acid position F 125 and aserine, arginine, or histidine substitution at amino acid position N24.11. The protein of claim 1, wherein the protein comprises the amino acidsequence shown in SEQ ID NO:2 with a leucine substitution at amino acidposition F125 and a serine substitution at amino acid position N24. 12.The protein of claim 1, wherein the protein comprises the amino acidsequence shown in SEQ ID NO:2 with a leucine or valine substitution atamino acid position F125, and a serine, arginine, or histidinesubstitution at amino acid position N24, and a lysine substitution atamino acid position M183.
 13. The protein of claim 1, wherein theprotein comprises the amino acid sequence shown in SEQ ID NO:2 with aleucine substitution at amino acid position F125, a serine substitutionat amino acid position N24, and a lysine substitution at amino acidposition M183.
 14. The protein of claim 1, wherein the protein comprisesthe amino acid sequence shown in SEQ ID NO:2 with a methioninesubstitution at amino acid position K164.
 15. The protein of claim 1,wherein the protein comprises the amino acid sequence shown in SEQ IDNO:2 with a leucine substitution at amino acid position F125.
 16. Anisolated Discosoma red fluorescent protein, the protein comprising theamino acid sequence shown in SEQ ID NO:2 with one or more pointmutations at an amino acid position selected from the group consistingof N24, F125, K164, and M183, and further comprising one or more pointmutations at an amino acid position selected from the group consistingof K93, R18, K139, E149 and D170.
 17. The protein of claim 16, whereinthe point mutation at amino acid position K93 is an argininesubstitution.
 18. The protein of claim 16, wherein the point mutation atamino acid position R18 is a histidine substitution.
 19. The protein ofclaim 16, wherein the point mutation at amino acid position E149 is anaspartic acid substitution.
 20. The protein of claim 16, wherein thepoint mutation at amino acid position D170 is a glycine substitution.21. The protein of claim 18, wherein the protein comprises the aminoacid sequence shown in SEQ ID NO:2 with a leucine substitution at aminoacid position F 125, a serine substitution at amino acid position N24, alysine substitution at amino acid position M183, and a histidinesubstitution at amino acid position R18.
 22. The protein of claim 19,wherein the protein comprises the amino acid sequence shown in SEQ IDNO:2 with a leucine substitution at amino acid position F125, a asparticacid substitution at amino acid position E149, and a glycinesubstitution at amino acid position D170.
 23. A fusion proteincomprising the protein of claim 1.